Therapeutic antibodies constitute the major category of biopharmaceuticals into the marketplace (Walsh G, 2014, Nature Biotechnology 2014, 32: 992-1000). Several therapeutic antibodies have obtained registration approval for the treatment of cancer, autoimmune diseases and other chronic diseases and dozens of recombinant antibodies are in different phases of clinical development (Biologic Medicines in Development, Phrma Report 2013, www.phrma.org). Usually patients receive hundreds milligrams of therapeutic antibodies in each dose, therefore there is currently a huge demand of production capacity worldwide.
Several approaches have been followed to increase the productivity of the industrial cell lines, for example, gene amplification systems, cell culture medium optimization and methods to select high producer cell clones. Genetic modification and epigenetic adaptation of recombinant myeloma cell lines to produce therapeutic antibodies is currently a very competitive research field (Barnes et al., 2000, Cytotechnology 32:109-123; Barnes et al., 2007, Biotechnol Bioeng 96:337-349).
Perfusion fermentation-based production processes allow high density cell cultures and potentially high antibody concentration in fermentation harvest. However long-term high density cell culture requires stable high producer clones to really optimize antibody production. Protein-free media have been developed for biopharmaceutical production, for example PFHMII cell culture medium (reference from Hyclone).
Recombinant antibody-producing NS0 cell clones have been successfully adapted to grow in protein-free media (WO 2004/038010 A1). However, adaptation to serum free-media and further long-term fermentation process in serum-free media are most usually accompanied by a loss of cell line productivity (Barnes et al., 2003, Biotechnol Bioeng 81: 631-639; Barnes et al., 2004, Biotechnol Bioeng 85: 115-121). Stable high producer cell clones adapted to grow in protein-free medium can be recovered from unstable recombinant myeloma cell lines for industrial use (CN104152415A).
But in practice for some recombinant myeloma cell lines the process of adaptation from serum-free medium to protein-free medium is not possible or takes long-time and is accompanied by the loss of antibody production due to the emergence of non-producer cell population. Innovation in the selection process of cell clones with industrial potential is warranted.
Biopharmaceuticals, in particular therapeutic antibodies, are complex glycoprotein molecules. Any change in the production process might introduce variations in product attributes. The concept of comparability has emerged to assess such variations in product attributes (Demonstration of comparability of human biological products, including therapeutic biotechnology-derived products, Center for Biologics Evaluation and Research (CBER), Center for Drug Evaluation and Research (CDER) April 1996. www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm122879.htm; EU Guideline on Comparability of Medicinal Products containing Biotechnology-derived Proteins as Active Substances: Quality issues (CPMP December 2003). www.emea.europa.eu/pdfs/human/bwp/320700en.pdf; ICH Q5E: Comparability of Biotechnological/Biological Products Subject to Changes in their Manufacturing Process. EU: Adopted by CMPM, Dec. 1, 2004, CPMP/ICH/5721/03, date for coming into operation: June 2005; MHLW: Adopted 26 Apr. 2005, PFSB/ELD Notification No. 0426001; FDA: Published in the Federal Register, Vol. 70, No. 125, Jun. 30, 2005; 37861-2 www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q5E/Step4/Q5E_Guideline.pdf). Change in the cell line is considered one the critical modification in the production process. Therefore although selection of cell lines with industrial potential is absolutely required in the production process development it is not obvious that any of the selected cell lines with the required characteristics of stability and high production rate will be suitable because variations in the secreted immunoglobulin might affect its biological properties. Definition of identity attributes of each specific antibody is a prerequisite for any further comparability studies related to production process modification, for example production scale or manufacturing site.
14F7 is a monoclonal antibody (mAb) specific for the tumor associated antigen N-glycolyl-GM3 ganglioside [GM3(Neu5Gc)] (Patent number ZL 99800261.5; Carr et al., 2000, Hybridoma 19, 241-247). This antigen has been detected in breast cancer (Marquina et al., 1996, Cancer Res 56, 5165-5171; Oliva et al., 2006, Breast Cancer Res Treat 96, 115-121), melanoma (Osorio et al., 2008, Cancer BiolTher 7, 488-495), non-small cell lung cancer (van Cruijsen et al., 2009, BMC Cancer 9, 180; Hayashi et al., 2013, Cancer Sci 104, 43-47), Wilms tumors (Scursoni et al., 2010, Pediatr Dev Pathol 13, 18-23), neuroectodermal tumors (Scursoni et al., 2011, Clin Dev Immunol 245181), sarcomas and thyroid carcinomas (Blanco et al., 2013, Journal of Biomarkers, 602417) and malignancies from the digestive (Blanco et al., 2011, ISRN Gastroenterol 645641) and genitourinary (Blanco et al., 2011, ISRN Pathology, 953803) systems.
14F7 mAb is able to kill tumor cells expressing the ganglioside in a complement-independent manner (Carr et al., 2002, Hybrid Hybridomics 21, 463-468; Roque-Navarro et al., 2008, Mol Cancer Ther 7, 2033-2041). A humanized version of this antibody, obtained by the modification of potential human T cell epitopes (Patent application No. 200480017457.8; Mateo et al., 2000, Hybridoma 19, 463-471) and named 14F7h, retained the properties of the mouse and chimeric antibodies (Fernandez-Marrero et al., 2011, Immunobiology 216, 1239-1247). Humanized 14F7hmAb is of potential value for the therapy of GM3(NeuGc)-expressing tumors. [0010] However recombinant NS0 myeloma cell lines expressing antibody 14F7h lost viability in high density cell cultures because these cells expressed the antigen GM3(Neu5Gc) and were killed by the secreted cytotoxic antibody. Therefore the recombinant humanized antibody 14F7h was expressed in murine NS0 myeloma cell line defective in N-glycolylated-glycoconjugates, due to the knock down of the CMP-N-acetylneuraminic acid hydroxylase enzyme (Fernandez-Marrero et al., 2011, Immunobiology 216, 1239-1247). Such cell line showed to be very difficult to adapt to grow in serum-free medium.
In the present invention we have established a new approach to develop stable high producer cell clones to be used in perfusion fermentation production process. Following this approach cell clones were recovered from a recombinant NS0 myeloma cell line which produces an anti-GM3(NeuGc) monoclonal antibody. Identity attributes of such therapeutic antibody are also disclosed in this invention, which define a molecular phenotype for this biopharmaceutical.